Thermolysin coordination number

In particular, in Cd(II)-Thermolysin derivative (a zinc metaUoprotease with proteolytic activity similar to carboxypeptidase A), the X-ray structure has provided evidence for isostructural replacement of Zn(II) by Cd(II). In general, the " Cd chemical shift is very sensitive to the nature, number and coordination type of the amino acid ligands and " Cd resonances are commonly detected by direct observation (/ = V2, and 63% sensitivity compared with C) or by inverse detection of Cd scalar-coupled to H. 

Another technique that uses the fluorescence properties of trivalent lanthanides is that of the detection of fluorescence emission decay induced by pulsed dye laser excitation. Horrocks and Sudnick (17) have applied this technique to the study of water molecules bound to metal ions in small complexes and proteins. In one study they found that the exponential decay of Tb3+ fluorescence is altered when H20 is replaced by D20 and that this change can be used to determine the number of coordinated water molecules on the metal ion. With thermolysin, bound Tb3+ had 1-2 water molecules in the first coordination shell. This number is consistent with the x-ray structure. 

In order to determine the number of histidine residues involved in zinc coordination, the L chains of TeTx and BoNT/A, B and E were modified with diethyl pyrocarbonate (DEPC), a reagent that specifically modifies histidine residues. In each case, two additional histidines were modified in the apo-toxin that were not affected in the holo-neurotoxin (Schiavo etal., 1992 b, c). These results indicate that the zinc atom of CNTs is coordinated via two histidines and a Glu-bound water molecule, as in thermolysin. Mutations at the two histidines of the motif inactivate TeTx and suppress its ability to bind radio-labeled Zn " (Yamasaki etal., 1994 b). In addition, mutations of the conserved Glu-271 and Glu-272 of TeTx, predicted to be in an a-helical segment (Lebeda and Olson, 1994), result in decreased zinc binding and loss of activity. Based on these experimental results, it has... 

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